Quinoline derivatives, process for preparing them and use for the treatment of diseases mediated by s-CD23

ABSTRACT

A compound of formula (I): useful in the treatment and prophylaxis of conditions mediated by s-CD23

This invention relates to novel inhibitors of the formation of solublehuman CD23 and their use in the treatment of conditions associated withexcess production of soluble CD23 (s-CD23) such as autoimmune diseaseand allergy.

CD23 (the low affinity IgE receptor FceRII, Blast 2), is a 45 kDa typeII integral protein expressed on the surface of a variety of maturecells, including B and T lymphocytes, macrophages, natural killer cells,Langerhans cells, monocytes and platelets (Delespesse et al, AdvImmunol, 49 [1991] 149–191). There is also a CD23-like molecule oneosinophils (Grangette et al, J Immunol, 143 [1989] 3580.3588). CD23 hasbeen implicated in the regulation of the immune response (Delespesse etal, Immunol Rev, 125 [1992] 77–97). Human CD23 exists as twodifferentially regulated isoforms, a and b, which differ only in theamino acids at the intracellular N-terminus (Yokota et al, Cell, 55[1988] 611–618). In man the constitutive a isoform is found only onB-lymphocytes, whereas type b, inducible by IL4, is found on all cellscapable of expressing CD23.

Intact, cell bound CD23 (i-CD23) is known to undergo cleavage from thecell surface leading to the formation of a number of well-definedsoluble fragments (s-CD23), which are produced as a result of a complexsequence of proteolytic events, the mechanism of which is still poorlyunderstood (Bourget et al J Biol Chem, 269 [1994] 6927–6930). Althoughnot yet proven, it is postulated that the major soluble fragments (Mr37, 33, 29 and 25 kDa) of these proteolytic events, all of which retainthe C-terminal lectin domain common to i-CD23, occur sequentially viainitial formation of the 37 kDa fragment (Letellier et al, J Exp Med,172 [1990] 693–700). An alternative intracellular cleavage pathway leadsto a stable 16 kDa fragment differing in the C-terminal domain fromi-CD23 (Grenier-Brosette et al, Eur J Immunol, 22 [1992] 1573–1577).

Several activities have been ascribed to membrane bound i-CD23 inhumans, all of which have been shown to play a role in IgE regulation.Particular activities include: a) antigen presentation, b) IgE mediatedeosinophil cytotoxicity, c) B cell homing to germinal centres of lymphnodes and spleen, and d) downregulation of IgE synthesis (Delespesse etal, Adv Immunol, 49, [1991] 149–191). The three higher molecular weightsoluble CD23 fragments (Mr 37, 33 and 29 kDa) have multifunctionalcytokine properties which appear to play a major role in IgE production.Thus, the excessive formation of s-CD23 has been implicated in theoverproduction of IgE, the hallmark of allergic diseases such asextrinsic asthma, rhinitis, allergic conjunctivitis, eczema, atopicdermatitis and anaphylaxis (Sutton and Gould, Nature, 366, [1993]421–428).

Other biological activities attributed to s-CD23 include the stimulationof B cell growth and the induction of the release of mediators frommonocytes. Thus, elevated levels of s-CD23 have been observed in theserum of patients having B-chronic lymphocytic leukaemia (Sarfati et al,Blood, 71 [1988] 94–98) and in the synovial fluids of patients withrheumatoid arthritis (Chomarat et al, Arthritis and Rheumatism, 36[1993] 234–242). That there is a role for CD23 in inflammation issuggested by a number of sources. First, sCD23 has been reported to bindto extracellular receptors which when activated are involved incell-mediated events of inflammation. Thus, sCD23 is reported todirectly activate monocyte TNF, IL-1, and IL-6 release (Annant et al,vol 180, J. Exp. Med., 1005–1011 (1994)). CD23 has been reported tointeract with the B2-integrin adhesion molecules, CD11b and CD11c onmonocyte/macrophage (S. Lecoanet-Henchoz et al, Immunity, vol 3; 119–125(1995)) which trigger NO2⁻, hydrogen peroxide and cytokine (IL-1, IL-6,and TNF) release. Finally, IL-4 or IFN induce the expression of CD23 andits release as sCD23 by human monocytes. Ligation of the membrane boundCD23 receptor with IgE/anti-IgE immune complexes or anti CD23 mAbactivates cAMP and IL-6 production and thromboxane B2 formation,demonstrating a receptor-mediated role of CD23 in inflammation.

Because of these various properties of CD23, compounds which inhibit theformation of s-CD23 should have twofold actions of a) enhancing negativefeedback inhibition of IgE synthesis by maintaining levels of i-CD23 onthe surface of B cells, and b) inhibiting the immunostimulatory cytokineactivities of higher molecular weight soluble fragments (Mr 37, 33 and29 kDa) of s-CD23. In addition, inhibition of CD23 cleavage shouldmitigate sCD23-induced monocyte activation and mediator formation,thereby reducing the inflammatory response.

WO 99/06361 (Abbott) and WO 00/12478 (Zeneca Limited) describe a rangeof compounds which includes reverse hydroxamate sulfonyl and sulfonamidecompounds, for use as metalloproteinase inhibitors.

WO 99/38843 (Darwin Discovery Limited) discloses a generic scope ofcompounds useful in the treatment of inter alia conditions mediated byenzymes involved in the shedding of CD23, which covers compounds of theformula (A):

wherein B, R¹ and R² are selected from a range of organic groups.

PCT EPO1/05798 (SmithKline Beecham p.l.c.) discloses compounds useful inthe treatment and prophylaxis of conditions mediated by enzymes involvedin the shedding of CD23 which covers compounds of formula (B):

Wherein R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl orheterocyclyl; and R¹ is bicyclyl or heterobicyclyl.

According to the present invention, there is provided a compound offormula (I):

According to a further aspect, the present invention provides the use ofa compound of formula (I) for the production of a medicament for thetreatment or prophylaxis of disorders such as allergy, allergic asthma,atopic dermatitis and other atopic diseases; inflammatory disorders; andautoimmune disease, in which the overproduction of s-CD23 is implicated.

In a further aspect the invention provides a method for the treatment orprophylaxis of disorders such as allergy, allergic asthma, atopicdermatitis and other atopic diseases; inflammatory disorders; andautoimmune disease, in which the overproduction of s-CD23 is implicated,which method comprises the administration of a compound of formula (I),to a human or non-human mammal in need thereof.

The invention also provides a pharmaceutical composition for thetreatment or prophylaxis of disorders such allergy, allergic asthma,atopic dermatitis and other atopic diseases; inflammatory disorders; andautoimmune disease, in which the overproduction of s-CD23 is implicatedwhich comprises a compound of formula (I) and optionally apharmaceutically acceptable carrier therefor.

Particular inflammatory disorders include CNS disorders such asAlzheimer's disease, multiple sclerosis, and multi-infarct dementia, aswell as the inflammation mediated sequel of stroke and head trauma.

The present inventors have surprisingly found that the compound of theinvention is a highly potent and selective inhibitor of CD23 processing,having little or no activity as an inhibitor of matrix metalloproteases.

It is to be understood that the pharmaceutically acceptable salts,solvates and other pharmaceutically acceptable derivatives of thecompound of formula (I) are also included in the present invention.

Salts of compounds of formula (I) include for example acid additionsalts derived from inorganic or organic acids, such as hydrochlorides,hydrobromides, hydroiodides, p-toluenesulphonates, phosphates,sulphates, acetates, trifluoroacetates, propionates, citrates, maleates,fumarates, malonates, succinates, lactates, oxalates, tartrates andbenzoates.

Salts may also be formed with bases. Such salts include salts derivedfrom inorganic or organic bases, for example alkali metal salts such assodium or potassium salts, and organic amine salts such as morpholine,piperidine, dimethylamine or diethylamine salts.

The compounds of the invention may be prepared by use of any appropriateconventional method.

In a further aspect the present invention provides a process forpreparing compounds of formula (I) as defined hereinabove, which processcomprises:

(a) deprotecting a compound of formula (II):

where P is a protecting group such as benzyl, tetrahydropyranyl orp-methoxybenzyl, or

(b) formylating a compound of formula (III):

or(c) oxidising a compound of formula (X):

Compounds of formula (II), (III) and (X) are novel and form a furtheraspect of the invention.

The following reaction schemes illustrate the procedures that may beused to prepare compounds of formula (I).

REACTION SCHEMES

One procedure for preparing compounds of formula (I) is shown inScheme 1. The thiol (VIII) may be prepared from the corresponding halidesuch as the bromide (IX) using the methods described by Choi and Yoon,Synthesis, 1995, 373, and converted into (VII) by reaction with asuitable halomethyl ketone such as the bromomethyl ketone in thepresence of a base such as triethylamine. The ketone (VII) can bereacted with a suitably O-protected hydroxylamine. For example, when theprotecting group (P) is benzyl, reaction with O-benzyl hydroxylamineunder standard conditions can be used to prepare oxime (VI) which can bereduced to (V) with a suitable reducing agent eg sodium borohydride orsodium cyanoborohydride in acetic acid. Formylation of (V) using formicacetic anhydride followed by oxidation with meta chloroperbenzoic acidaffords (II) which can be deprotected under suitable conditions.

Compounds of formula (I) may also be prepared as described in Scheme 2.The ketone (VII) may be reacted with hydroxylamine under standardconditions to give an oxime (XII) which upon reduction with a suitablereducing agent such as sodium cyanoborohydride in acetic acid yields thehydroxylamine (XI) which can be formylated by treatment with formicacetic anhydride followed by potassium carbonate and methanol, andoxidised as described previously for Scheme 1.

The compound of the present invention may be prepared as a mixture ofisomers or as an individual isomer. The individual isomer may beprepared by any appropriate method, for example individual stereoisomersmay be prepared by stereospecific chemical synthesis starting fromchiral substrates or by separating mixtures of enantiomers or mixturesof diastereomers using known methods such as chiral preparative HPLC.For example, separation of compounds of formula (I) which are racemicinto single enantiomers can be achieved by conversion into a suitableester derivative such as the O-methyl mandelic acid derivative followedby separation using standard chromatographic procedures and thendeprotection.

In a preferred aspect, the invention provides a compound of formula(IA):

Compounds of formula (I) can be prepared in chirally pure form using theprocedures described in Scheme 3. In the preparation of compounds offormula (IA), for example, a suitably protected chiral amino alcohol(XIXA) can be converted into the corresponding thioacetate (XVIIIA)under Mitsunobu conditions eg using triphenylphosphine ortributylphosphine in combination with di-t-butylazodicarboxylate ordiethylazodicarboxylate. The thioacetate (XVIIIA) can be converted insitu into a thiol and thence into the sulfide (XVIIA). Alternatively,the alcohol group in (XIXA) may be converted into a leaving group suchas a tosylate or bromide and reacted with the appropriate thioacetate orthiol, such as quinolin-3-yl-methanethiol or thioacetate, in thepressence of a suitable base such as sodium hydroxide to give (XVIIA).Oxidation of (XVIIA) to the sulfone (XVIA) can be carried out with asuitable oxidising agent such as MCPBA, followed by deprotection.Deprotection under standard conditions such as trifluoroacetic acid orhydrogen chloride in dioxan affords (XVA) which can be converted into(XIVA) and oxidised to the oxaziridine (XIIIA) using establishedprocedures eg meta chloroperbenzoic acid. Conversion of the oxaziridine(XIIIA) into (IIIA) is preferably carried out using hydrochloric acid.Formylation of (IIIA) can be carried out using standard formylationmethodology such as reaction with formic-acetic anhydride.

An alternative procedure for preparing compounds of formula (IIIA) inchirally pure form is shown in Scheme 4. The chiral amine (XVA) can bealkylated with cyanomethyl bromide or cyanomethyl iodide to give thecyanomethylamine (XXA) which is converted into (IIIA) using the methodsdescribed by Tokuyama et al., Synthesis, 2000, 9, 1299.

Alternatively, the amine (XVA) may be oxidised directly with benzoylperoxide to give a benzoyl hydroxylamine (XXIA) as shown in Scheme 5,using the method described by Phanstiel, J. Org. Chem., 1997, 62,8104.The latter compound can be formylated with formic acetic anhydride andthen deprotected, for example with ammonia in methanol, to give (IA).

Compounds of formula (III) can also be prepared using the route shown inScheme 6. The alcohol (XXIV) can be obtained by reduction of the ketone(VII) under standard conditions, for example borane in THF. Whereappropriate, the alcohol (XXIV) may be prepared by reaction of the haloalcohol (XXVI) with (XXV). Oxidation to the sulfone (XXIII) can becarried out as previously described followed by elimination usingmethanesulfonyl chloride and triethylamine, or Mitsunobu conditions togive (XXII). Addition of hydroxylamine to the unsaturated sulfone (XXII)affords (III).

Halomethyl ketones can be obtained by bromination of a methyl ketoneusing bromine in methanol as described by Gaudry and Marquet, Org.Synth., 1976, 55, 24. Alternatively, bromomethylketones can be obtainedfrom the corresponding diazoketones by reaction with hydrogen bromideusing standard methods. Compounds of formula (IX) can be obtained bystandard methods, for example by bromination of 3-methyl-quinoline withN-bromosuccinimide in carbon tetrachloride. Bromination of quinolinecontaining precursors with N-bromosuccinimide is preferably carried outin the presence of an acid such as acetic acid. Alternativelyquinolin-3-yl-methanol may be converted into a compound of formula (IX)using standard halogenation procedures e.g. using phosphorouspentachloride or thionyl chloride, or by conversion into the mesylatefollowed by treatment with lithium bromide in acetone.

Protected amino alcohol precursors are either commercially available ormay be prepared via standard routes e.g. from the corresponding aminoacids as described by Ho et al, Tet. Lett., 1993, 34(41), 6513.

Suitable amino acid derivatives may be prepared from aziridineprecursors e.g. as described by Nakajima et al., Bull. Soc. Chim. Japan,1982, 55, 3049.

The other starting materials and other reagents are availablecommercially or can be synthesised by well-known and conventionalmethods.

It is preferred that the compound is isolated in substantially pureform.

As stated herein an inhibitor of the formation of soluble human CD23 hasuseful medical properties. Preferably the active compound isadministered as pharmaceutically acceptable compositions.

The compositions are preferably adapted for oral administration.However, they may be adapted for other modes of administration, forexample in the form of a spray, aerosol or other conventional method forinhalation, for treating respiratory tract disorders; or parenteraladministration for patients suffering from heart failure. Otheralternative modes of administration include sublingual or transdermaladministration.

The compositions may be in the form of tablets, capsules, powders,granules, lozenges, suppositories, reconstitutable powders, or liquidpreparations, such as oral or sterile parenteral solutions orsuspensions.

In order to obtain consistency of administration it is preferred that acomposition of the invention is in the form of a unit dose.

Unit dose presentation forms for oral administration may be tablets andcapsules and may contain conventional excipients such as binding agents,for example syrup, acacia, gelatin, sorbitol, tragacanth, orpolyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch,calcium phosphate, sorbitol or glycine; tabletting lubricants, forexample magnesium stearate; disintegrants, for example starch,polyvinylpyrrolidone, sodium starch glycollate or microcrystallinecellulose; or pharmaceutically acceptable wetting agents such as sodiumlauryl sulphate.

The solid oral compositions may be prepared by conventional methods ofblending, filling or tabletting. Repeated blending operations may beused to distribute the active agent throughout those compositionsemploying large quantities of fillers. Such operations are of courseconventional in the art. The tablets may be coated according to methodswell known in normal pharmaceutical practice, in particular with anenteric coating.

Oral liquid preparations may be in the form of, for example, emulsions,syrups, or elixirs, or may be presented as a dry product forreconstitution with water or other suitable vehicle before use. Suchliquid preparations may contain conventional additives such assuspending agents, for example sorbitol, syrup, methyl cellulose,gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminiumstearate gel, hydrogenated edible fats; emulsifying agents, for examplelecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (whichmay include edible oils), for example almond oil, fractionated coconutoil, oily esters such as esters of glycerine, propylene glycol, or ethylalcohol; preservatives, for example methyl or propyl p-hydroxybenzoateor sorbic acid; and if desired conventional flavouring orcolouring-agents.

For parenteral administration, fluid unit dosage forms are preparedutilising the compound and a sterile vehicle, and, depending on theconcentration used, can be either suspended or dissolved in the vehicle.In preparing solutions the compound can be dissolved in water forinjection and filter sterilised before filling into a suitable vial orampoule and sealing. Advantageously, adjuvants such as a localanaesthetic, a preservative and buffering agents can be dissolved in thevehicle. To enhance the stability, the composition can be frozen afterfilling into the vial and the water removed under vacuum. Parenteralsuspensions are prepared in substantially the same manner, except thatthe compound is suspended in the vehicle instead of being dissolved, andsterilisation cannot be accomplished by filtration. The compound can besterilised by exposure to ethylene oxide before suspending in thesterile vehicle. Advantageously, a surfactant or wetting agent isincluded in the composition to facilitate uniform distribution of thecompound.

Compositions of this invention may also suitably be presented foradministration to the respiratory tract as a snuff or an aerosol orsolution for a nebulizer, or as a microfine powder for insufflation,alone or in combination with an inert carrier such as lactose. In such acase the particles of active compound suitably have diameters of lessthan 50 microns, preferably less than 10 microns for example diametersin the range of 1–50 microns, 1–10 microns or 1–5 microns. Whereappropriate, small amounts of other anti-asthmatics and bronchodilators,for example sympathomimetic amines such as isoprenaline, isoetharine,salbutamol, phenylephrine and ephedrine; xanthine derivatives such astheophylline and aminophylline and corticosteroids such as prednisoloneand adrenal stimulants such as ACTH may be included.

The compositions may contain from 0.1% to 99% by weight, preferably from10–60% by weight, of the active material, depending upon the method ofadministration. A preferred range for inhaled administration is 10–99%,especially 60–99%, for example 90, 95 or 99%.

Microfine powder formulations may suitably be administered in an aerosolas a metered dose or by means of a suitable breath-activated device.

Suitable metered dose aerosol formulations comprise conventionalpropellants, cosolvents, such as ethanol, surfactants such as oleylalcohol, lubricants such as oleyl alcohol, desiccants such as calciumsulphate and density modifiers such as sodium chloride.

Suitable solutions for a nebulizer are isotonic sterilised solutions,optionally buffered, at for example between pH 4–7, containing up to 20mg/ml of compound but more generally 0.1 to 10 mg/ml, for use withstandard nebulisation equipment.

An effective amount will depend on the relative efficacy of thecompounds of the present invention, the severity of the disorder beingtreated and the weight of the sufferer. Suitably, a unit dose form of acomposition of the invention may contain from 0.1 to 1000 mg of acompound of the invention (0.001 to 10 mg via inhalation) and moreusually from 1 to 500 mg, for example 1 to 25 or 5 to 500 mg. Suchcompositions may be administered from 1 to 6 times a day, more usuallyfrom 2 to 4 times a day, in a manner such that the daily dose is from 1mg to 1 g for a 70 kg human adult and more particularly from 5 to 500mg. That is in the range of about 1.4×10 m2 mg/kg/day to 14 mg/kg/dayand more particularly in the range of about 7×10–2 mg/kg/day to 7mg/kg/day.

The following example illustrates the invention but does not limit it inany way.

Preparation 1: 3-Acetylthiomethylquinoline

Method A

Step 1: 3-Quinolylmethanol—Quinoline-3-carboxaldehyde (13.18 g) inethanol (260 ml) was cooled to 0° C. followed by the addition of sodiumborohydride (1.62 g) portionwise. The temperature was maintained at 0°C. for 15 min followed by the addition of 6N HCl (28 ml) during whichtime the temperature of the reaction was maintained between 0–5° C. Thesolution was then neutralised with 1M NaOH. The crude reaction mixturewas stripped to dryness to remove ethanol and the residue waspartitioned between water and EtOAc. The EtOAc layer was then dried(MgSO₄) and absorbed onto silica gel and chromatographed (flash silicagel, step gradient: 0–100% EtOAc/hexane) to give the subtitle compoundas a white solid (9.85 g).

Step 2: 3-Chloromethylquinoline hydrochloride—3-Quinolylmethanol (9.85g) was taken up in dry benzene (200 ml) and stirred followed by theaddition of thionyl chloride (14.69 ml). An immediate yellow precipitatewas obtained. Stirring was maintained at rt for 2 h. A light yellowsolid was filtered off and dried to give the subtitle compound (13 g).

Step 3: 3-Acetylthiomethylquinoline—3-Chloromethylquinolinehydrochloride (5.2 g) was taken up in acetone (100 ml) followed by theaddition of potassium thioacetate (1.8 g) and allowed to stir at rtovernight. The reaction mixture was absorbed onto silica gel andchromatographed (silica gel, step gradient 0–50% ether/petroleum ether)to give the title compound as an orange solid (4.2 g). ¹H NMRδ(DMSO-d6): 8.85 (1H, d, J=2 Hz), 8.25(1H, d, J=2 Hz), 8.01(1H, d, J=8.4Hz), 7.95 (1H, d, J=8.4 Hz), 7.74 (1H, t, J=8.4 Hz), 7.61 (1H, t, J=8.4Hz), 4.33 (2H, s), 2.38 (3H, s).

Method B

3-Methylquinoline (5 g) in CCl₄ (50 ml) was treated with glacial aceticacid (1.85 ml), NBS (8.5 g) and AIBN (1.5 g). The reaction was broughtto reflux using a 100W halogen light and refluxed for 10 min. Aftercooling, EtOAc (60 ml) was added and the reaction was filtered through aplug of silica, concentrated to half volume and added to potassiumthioacetate (10 g) dissolved in DMF (150 ml) with potassium carbonate (2g). The reaction was then further concentrated to 150 ml by evaporation.After 2 h the reaction was diluted with EtOAc (300 ml) and washed withsaturated sodium hydrogen carbonate solution and saturated brine (8×).The organic phase was evaporated and the residue chromatographed (silicagel, step gradient 0–50% ether/petroleum ether) to afford the titlecompound (3.1 g).

EXAMPLE 1(S)-N-[2-(3-Quinolylmethanesulfonyl)-1-(R)-tetrahydrofuran-2-ylethyl]-N-hydroxyformamide

Step 1:1-(3-quinolylmethanesulfanyl)-1-[(R)-tetrahydrofuran-2-yl]-1-oxoethane—Asolution of 3-acetylthiomethylquinoline (3.15 g) in MeOH (50 ml) wastreated with a 1M solution of NaOH in MeOH for 15 min followed by theaddition of (R)-2-bromoacetyltetrahydrofuran (2.8 g) (European patent540609-A1). The reaction mixture was stirred at rt overnight. Themixture was evaporated to low volume, diluted with EtOAc (50 ml), washedwith saturated sodium hydrogen carbonate solution (30 ml), water (2×30ml), saturated brine (30 ml), dried (MgSO₄) and evaporated. The residuewas flash chromatographed. (silica gel, step gradient: 30–50%EtOAc/hexane) to give the subtitle compound (4.16 g). MS APCI (+ve ion)288 (MH⁺); ¹H NMR δ(CDCl3): 8.88 (1H, d, J=2 Hz), 8.09(H, m), 7.76 (1H,d, J=8.3 Hz), 7.70 (1H, t, J=8.3 Hz), 7.54(1H, t, J=7 Hz), 4.45 (1H,m),4.20–3.60 (2H,m), 3.91 (2H, s), 3.30 (2H, s), 2.25–1.83 (4H,m).

Step 2:(RS)-2-(3-quinolylmethanesulfanyl)-1-[(R)-tetrahydrofuran-2-yl]-1-hydroxyethane—Anice-cold solution of1-(3-quinolyhnethanesulfanyl)-1-[(R)-tetrahydrofuran-2-yl]-1-oxoethane(1.52 g) in MeOH (30 ml) was treated with sodium borohydride (0.2 g).After 20 minutes the solution was evaporated and redissolved in EtOAc(30 ml) and saturated sodium hydrogen carbonate solution (20 ml). Theorganic phase was collected washed with water (20 ml), saturated brine(20 ml), dried (MgSO₄) and evaporated. The residue was flashchromatographed. (silica gel, step gradient:50–100% EtOAc/hexane) togive the subtitle compound (1.22 g).MS APCI (+ve ion) 290 MH⁺, ¹H NMRδ(CDCl3) 8.90 (1H,d,J=2 Hz), 8.08 (2H,m),7.76 (1H,d,J=8.3 Hz),7.70(1H,t,J=8.3 Hz), 7.54 (1H,t,J=7 Hz), 4.05–3.50 (5H,m),2.60 (3H,m), 1.85(4H,m).

Step 3:2-(3-quinolylmethanesulfonyl)-1-[(R)-tetrahydrofuran-2-yl]-(RS)-1-hydroxyethane—Asolution of(RS)-2-(3-quinolylmethanesulfanyl)-1-[(R)-tetrahydrofuran-2-yl]-1-hydroxyethane(1.21 g) in dry MDC (20 ml) was cooled to 0° C. followed by the additionof MCPBA (50%) (2.89 g) and allowed to stir at 0° C. for 30 min. Thereaction mixture was quenched with 10% Na₂SO₃ (10 ml) and saturatedsodium hydrogen carbonate (10 ml). The MDC layer was dried (MgSO₄),evaporated to give the subtitle compound. (1.27 g). MS APCI (+ve ion)322 (MH⁺), ¹H NMR δCDCl₃ 8.88 (1H, d, J=2.4 Hz), 8.32 (1H, d, J=2.4 Hz),8.11 (1H, d, J=8.4 Hz), 7.86 (1H, d, J=8.4 Hz), 7.78 (1H, t, J=8.4 Hz),7.672(1H, t, J=8 Hz), 4.78–4.08 (3H, m), 3.82 (3H, m), 3.03 (2H, m),2.05–1.62 (4H, m)

Step 4:(E)-2-(3-Quinolylmethanesulfonyl)-1-(R)-tetrahydrofuran-2-ylethene—Anice-cold solution of2-(3-quinolylmethanesulfonyl)-1-[(R)-tetrahydrofuran-2-yl]-(RS)-1-hydroxyethane(1.26 g) in dry MDC (50 ml) was treated with pyridine (0.48 ml),4-dimethylaminopyridine (5 mg) and methanesulfonyl chloride (0.34 ml).After 15 minutes the mixture was allowed to gain room temperature andEt₃N (1.64 ml) added. After 15 minutes the mixture was washed with water(2×30 ml), saturated brine (30 ml), dried (MgSO₄) and evaporated. Theresidue was flash chromatographed (silica gel, step gradient: 60–100%EtOAc/hexane) to give the subtitle compound as a white solid (0.87 g).MS APCI (+ve ion) 304 (MH⁺), ¹H NMR δ(CDCl₃): 8.81 (1H, d, J=2 Hz), 8.31(1H, d, J=2 Hz), 8.06 (1H, d, J=8.4 Hz), 7.85 (1H, d, J=8.4 Hz), 7.74(1H, t, J=8.4 Hz), 7.60- (1H, t, J=8 Hz), 6.72 (1H, d, J=15 Hz), 6.50(1H, d,J=15 Hz), 4.51 (H, m), 4.41 (2H,s), 4.82 (2H,m), 2.18–1.41 (4H,m).

Step 5:N-[2-(3-Quinolylmethanesulfonyl)-1-(R)-tetrahydrofuran-2-yl]ethylhydroxylamine—Asolution of(E)-2-(3-Quinolylmethanesulfonyl)-1-(R)-tetrahydrofuran-2-ylethene (0.86g) in THF (10 ml) was treated with hydroxylamine (50 wt % solution inwater, 5 ml) and allowed to stir at rt for 15 minutes. The solution wasevaporated and then azeotroped first with MeOH and then EtOH/toluene togive the subtitle compound (952 mg). MS APCI (+ve ion) 337(MH⁺).

Step 6:(S)-N-[2-(3-Quinolylmethanesulfonyl)-1-(R)tetrahydrofuran-2-ylethyl]-N-hydroxyformamide-N-[2-(3-Quinolylmethanesulfonyl)-1-(R)-tetrahydrofuran-2-yl]ethylhydroxylamine(0.94 g) was treated with formic acid (6 ml) and acetic anhydride (2 ml)and stood overnight at rt. The reaction mixture was evaporated,redissolved in methanol and treated with K₂CO₃ (1.9 g). After stirringat rt for 30 min the mixture was evaporated and the residue waspartitioned between MDC and water and the pH adjusted to 7 (2M HCl). TheMDC layer was dried (MgSO₄) and evaporated to give a crude mixture ofdiastereomers which was separated into single diastereomers bychromatography. (Acid washed silica gel, Step gradient: 0–3% MeOH inMDC.) The slower running component was collected and recrystallised togive the title compound as a white solid (160 mg). MS APCI (+ve ion) 365(MH⁺), ¹H NMR δ(DMSO-d6) at 353K 9.756(1H,bs), 8.88 (1H, d,J=2 Hz), 8.35(1H, d,J=2 Hz), 7.88–8.30 (3H, m), 7.78 (1H, t, J=7.2 Hz), 7.66 (1H, t,J=7.2 Hz), 4.80–4.68 (2H, ABq,) 3.95(1H, m), 3.10–3.82 (3H, m),1.50–2.03(4H, m).

Biological Test Methods

Procedure 1: The ability of test compounds to inhibit the release ofsoluble CD23 was investigated by use of the following procedure.

RPMI 8866 Cell Membrane CD23 Cleavage Activity Assay:

Plasma membranes from RPMI 8866 cells, a human Epstein-Barr virustransformed B-cell line (Sarfati et al., Immunology 60 [1987] 539–547)expressing high levels of CD23 are purified using an aqueous extractionmethod. Cells resuspended in homogenization buffer (20 mM HEPES pH 7.4,150 mM NaCl, 1.5 mM MgCl2, 1 mM DTT) are broken by N₂ cavitation in aParr bomb and the plasma membrane fraction mixed with other membranes isrecovered by centrifugation at 10,000×g. The light pellet is resuspendedin 0.2 M potassium phosphate, pH 7.2 using 2 ml per 1–3 g wet cells andthe nuclear pellet is discarded. The membranes are further fractionatedby partitioning between Dextran 500 (6.4% w/w) and polyethylene glycol(PEG) 5000 (6.4% w/w) (ref), at 0.25 M sucrose in a total of 16 g per10–15 mg membrane proteins [Morre and Morre, BioTechniques 7, 946–957(1989)]. The phases are separated by brief centrifugation at 1000×g andthe PEG (upper) phase is collected, diluted 3–5 fold with 20 mMpotassium phosphate buffer pH 7.4, and centrifuged at 100,000×g torecover membranes in that phase. The pellet is resuspended inphosphate-buffered saline and consists of 3–4 fold enriched plasmamembranes as well as some other cell membranes (e.g. lysosomes, Golgi).The membranes are aliquoted and stored at −80° C. Fractionation at 6.6%Dextran/PEG yields plasma membranes enriched 10-fold.

The fractionated membranes are incubated at 37° C. for times up to 4 hrsto produce fragments of CD23 which are separated from the membrane byfiltration in 0.2 micron Durapore filter plates (Millipore) afterquenching the assay with a non-selecitve MMP inhibitor, e.g. 5 uMPreparation 1 from WO 95/31457([4-(N-Hydroxyamino)-2-(R)-isobutyl-3-(S)-(2-thiophenethiomethyl)succinyl]-(S)-phenylalanine-N-methylamidesodium salt, prepared according to the procedure described in Example 11of WO 90/05719). sCD23 released from the membrane is determined usingthe EIA kit from The Binding Site (Birmingham, UK) or a similar oneutilising MHM6 anti-CD23 mAb [Rowe et al., Int. J. Cancer, 29, 373–382(1982)] or another anti-CD23 mAb as the capture antibody in a sandwichEIA. The amount of soluble CD23 made by 0.5 ug membrane protein in atotal volume of 50 ul phosphate-buffered saline is measured by EIA andcompared to the amount made in the presence of various concentrations ofinhibitors. Inhibitors are prepared in solutions of water ordimethylsulfoxide (DMSO) and the final DMSO concentration is not morethan 2%. IC50's are determined by curve fitting as the concentrationwhere 50% inhibition of production of sCD23 is observed relative to thedifference in sCD23 between controls incubated without inhibitor.

Results

The compound of Example 1 showed an IC₅₀ value of 0.06 uM

Procedure 2: The ability of test compounds to inhibit matrixmetalloproteases was investigated using the following procedures.

Collagenase Inhibition Assay:

The potency of compounds to act as inhibitors of collagenase wasdetermined by the method of Cawston and Barrett (Anal. Biochem. 99,340–345, 1979), hereby incorporated by reference, whereby a 1 mMsolution of the inhibitor being tested or dilutions thereof, wasincubated at 37° C. for 18 h with collagen and human recombinantcollagenase, from synovial fibroblasts cloned, expressed and purifiedfrom E. Coli, (buffered with 150 mM Tris, pH 7.6, containing 15 mMcalcium chloride, 0.05% Brij 35, 200 mM sodium chloride and 0.02% sodiumazide). The collagen was acetylated ³H type 1 bovine collagen preparedby the method of Cawston and Murphy (methods in Enzymology 80, 711,1981)The samples were centrifuged to sediment undigested collagen and analiquot of the radioactive supernatant removed for assay on ascintillation counter as a measure of hydrolysis. The collagenaseactivity in the presence of 1 mM inhibitor, or dilution thereof, wascompared to activity in a control devoid of inhibitor and the resultsreported as that concentration effecting 50% of the collagenase (IC₅₀).

Results

The compound of Example 1 showed an IC₅₀ value of 100 uM in procedure 2.

General MMP Inhibition Assays:

Inhibition of matrix metalloprotease activity was determined using afluorescence quench assay with appropriate substrate. For example, MMPactivity was determined using MMP activated using trypsin, according toLark et al, Connective Tissue Res. 25, 52 (1990). The MMP is incubatedat room temperature in a microtitre plate in a total volume of 100 ul,containing 0.15 M Tris Cl, 15 mM CaCl2, 0.2 M NaCl, pH 7.6 (assaybuffer); inhibitor at concentrations up to 100 uM, with no more than 2%DMSO final concentration, 10 uM substrate (such as SDP-3815-PI forMMP-1, Peptides International). The MMP concentration is <10 nM, anddetermined empirically with the appropriate substrate to give at least a20-fold increase in fluorescence emission in 30 min. Fluorescentexcitation wavelength was 355 nm, emission wavelength 400–460 nm, anddata points are collected to generate slope (change in fluorescence withtime). Percent inhibition for each concentration is calculated from theslope at time zero, and IC50 values from the concentration dependence.MMP-1, 2, 3, 7, 9, 13, 14 may all be assayed in the same manner, usingcommercially available substrates reported to be effective for eachenzyme. Enzymes were obtained from Calbiochem and activated using thesame trypsin method.

Results

The compound of Example 1 showed an IC₅₀ value of >100 uM vs MMP-3, and6.0 uM vs MMP-13.

Abbreviations

-   Bn—Benzyl-   EtOAc—ethyl acetate-   h—hour-   min—minutes-   MCPBA—meta chloroperoxybenzoic acid-   MDC—dichloromethane-   rt—room temperature-   THF—tetrahydrofuran

1. A compound of formula (I):


2. A compound of formula (IA):


3. A pharmaceutical composition which comprises a compound according toclaim 1 and optionally a pharmaceutically acceptable carrier therefor.4. A process for preparing a compound according to claim 1 which processcomprises: (a) deprotecting a compound of formula (II):

wherein P is a protecting group selected from the group consisting ofbenzyl, tetrahydropyranyl and p-methoxybenzyl, or (b) formylating acompound of formula (III):

or (c) oxidising a compound of formula (X):


5. A compound of formula (II):

wherein P is a protecting group selected from the group consisting ofbenzyl, tetrahydropyranyl and p-methoxybenzyl.
 6. A compound of formula(III):


7. A compound of formula (X):